The present invention relates to an apparatus for performing bio-assays and more particularly to a disposable structure for performing polymerase chain reaction (PCR) and capillary electrophoresis (CE) in a protocol for specific bacterial detection and/or identification.
The presence of certain suspected spoilage organisms, pathogens, beneficial organisms, or any bacterium in a sample can be detected by performing nucleic acid (DNA) analysis. Different types of organisms have different DNA sequences, which are sources of genetic information. In any given sample, the different nucleotide sequences of the organisms present in the mixture form together a large, indistinguishable background of nucleic acid. If a suspected organism is a part of this mixture, a technique can be performed that will exponentially amplify the number of its specific DNA sequences relative to the others in the sample. A second technique can then be used to separate and measure the amount of each of the different substances relative to each other. If one substance is predominant relative to all of the other substances in the sample, the presence of the suspected organism is then confirmed.
DNA sequences are formed as chains, or strands of a double helix, which pair with each other in a very precise way to form complementary sequences. There are four nucleotide bases, or building blocks of DNA, which are: adenine, cytosine, guanine, and thymine, which are represented respectively by: A, C, G, and T. The xe2x80x9cAxe2x80x9d on one strand will always pair with the xe2x80x9cTxe2x80x9d on the other, and the xe2x80x9cCxe2x80x9d will always pair with the xe2x80x9cG,xe2x80x9d such that the strands are complementary. A gene""s sequence is the arrangement of these four letters as a sentence, which can be hundreds or thousands of characters long.
Polymerase chain reaction (PCR) is a technique for amplifying, or copying, the xe2x80x9ctargetxe2x80x9d DNA sequences of a pre-selected organism in a sample, possibly by a factor of several million. Copying a DNA sequence requires a supply of the four nucleotide bases, xe2x80x9cprimers,xe2x80x9d and DNA polymerase. The primers are short sequences of the beginning and ending portions of the two complementary xe2x80x9ctargetxe2x80x9d DNA sequences of an organism to be amplified. DNA polymerase is an enzyme that utilizes the primers and the nucleotide bases to form copies.
The first step of PCR is to heat the reaction mixture containing the target DNA, a large excess of primers, the four nucleotide bases, and DNA polymerase, such that the paired strands of all of the DNA in the sample denature, or separate. The single strands are now accessible for the primers. Next, the sample is cooled to allow double-strands to form again. Because of the large excess of primers, the two strands of the unbound, target DNA sequence templates bind to the complementary primers instead of with each other. In the third step, the temperature is adjusted to obtain maximum activity for the DNA polymerase enzyme. For each DNA sequence that is bound to a short primer, the enzyme will extend the primer""s sequence by xe2x80x9chookingxe2x80x9d letters together to be complementary to the remaining unmarked portions of the single strands, such that the original double helix for the DNA sequence is replicated. In other words, if after the primer, a single strand contains an xe2x80x9cAxe2x80x9d nucleotide, the enzyme adds a xe2x80x9cTxe2x80x9d to the end of primer that is bound to the strand. If the strand next contains a xe2x80x9cG,xe2x80x9d the enzyme adds a xe2x80x9cCxe2x80x9d to the new chain, and so on, until the end of the DNA strand. This process doubles the number of DNA double helix sequences for the xe2x80x9ctargetxe2x80x9d organism in the sample. PCR thus allows a tester to multiply unique regions of DNA so that they can be detected in large genomes. The PCR process can be repeated many times within a short period to exponentially amplify the DNA of the xe2x80x9ctargetxe2x80x9d organism in the sample.
Capillary electrophoresis (CE) is a technique for detecting whether a unique region of DNA has been amplified in comparison with other DNA in a sample. A measured quantity of the sample is introduced, with pressure or by applying a voltage (electrokinetic injection), into a sieving buffer-filled capillary. A voltage is then applied along the capillary, which causes the components in the sample to begin to move under the influence of the electrical field. Different components will move at different velocities, such that a separation can be made. The separated components then pass through a beam of light. Each component in the sample absorbs light of a given wavelength as it passes through the beam, and in response thereto, emits light of a different wavelength according to the fluorescence of the component. The light emitted by each component in response to the incident light is detected by a fluorometer as a series of peaks. The area of each peak is proportional to the amount of the substance present in the sample. A single dominant peak for a sample that has undergone PCR is indicative that the xe2x80x9ctargetxe2x80x9d DNA is present in the sample and was consequently amplified. Capillary electrophoresis is also useful when testing for a series of peaks in order to detect the presence of multiple products. As an example, this technique is used for performing a fingerprinting technology known as random amplified polymorphic DNA (xe2x80x9cRAPDxe2x80x9d).
Polymerase chain reaction and capillary electrophoresis are generally performed separately. PCR is typically performed in a reaction tube, and CE is performed in fused silica capillaries. Recently, it has been found that small samples may be processed in micro-devices, or microchips. It is known to fabricate devices for performing polymerase chain reaction and devices for performing capillary electrophoresis from crystalline semiconductor substrates. An advantage of using crystalline devices is that the construction can be precisely controlled through etching, and crystalline materials can be bonded by fusion at elevated temperatures. However, it is relatively expensive to fabricate PCR or CE devices from crystalline materials. Various fabrication techniques for plastic devices in CE also have been described including laser ablation, injection molding, silicone molding, and imprinting. However, these techniques were described for performing CE only.
Recently, it has been proposed to perform both polymerase chain reaction and capillary electrophoresis using a single, integrated device. The integration of PCR and CE using a single device allows for a substantial increase in operational speed. Because the process inherently involves less manipulation and handling when performed on a single chip, less operator skill is required. As a further benefit, the reduction of handling reduces problems of contamination and error.
There are many difficulties associated with designing a microchip for performing both PCR and CE. The medium selected must allow for the rapid thermal cycling that is required when heating and cooling a chamber for performing PCR. A material that is sufficiently thick to ensure rigidity and provide room for process elements may not facilitate good and rapid heat transfer needed for PCR. In contrast, it is counterproductive to cycle the temperature of the medium used for electrophoresis. The temperature changes to the electrophoresis portion of the device cause bubbles to form in the separation chamber, which can render the results of electrophoresis inconclusive.
In addition, many benefits of integrating PCR and CE into a single device cannot be realized unless the microchip is sufficiently inexpensive so as to be disposable. Further, a disposable PCR/CE system obviates the difficulties encountered in sterilizing the device for subsequent uses. Carry-over of only a few PCR product molecules into a subsequent PCR procedure can lead to the generation of false positive results. However, devices made from crystalline semiconductor substrates, glass, or a hybrid of both semiconductor substrates and glass, are too costly to be disposable.
In view of the various difficulties associated with performing PCR and CE in the conventional microdevices described above, there is a need for a device and a method for efficiently performing PCR and CE with accurate results, configured for use by an operator having a minimum of operator skill.
In accordance with the present invention, PCR and CE can be performed in a single, integrated apparatus, formed of a polymeric material, that is to be disposed after each use.
The integrated device of the present invention allows for localized heating of a PCR pouch and isolates the PCR pouch from the areas used for performing CE, thereby preventing the formation of bubbles in the solutions used for CE. The PCR pouch is formed by a cavity within a thin tab, surrounded by sealants, which allows for rapid thermal cycling for performing PCR. The device selectively allows or prevents fluid from reaching a first sample well of the CE device, by connecting or disassociating the PCR portion of the device from the CE portion of the device with a removable barrier. A vent channel is connected with the first sample well to allow air to escape.
In accordance with the invention, there is provided an apparatus for performing both polymerase chain reaction and capillary electrophoresis for a single sample in a single, disposable device that is to be discarded after a single use. The apparatus includes a polymerase chain reaction chamber, an electrophoresis channel, and a removable barrier between the polymerase chain reaction chamber and the electrophoresis channel.
In accordance with a further aspect of the present invention, there is provided an apparatus for performing polymerase chain reaction and capillary electrophoresis within a single, integrated and disposable device. The apparatus has a sample charging port by which a sample enters into the device. A polymerase chain reaction chamber is formed in a thin tab extended portion of the device, the chamber being defined by a cavity within the thin tab extension. An inflow channel is in fluid communication between the sample charging port and the polymerase chain reaction chamber. The apparatus also includes a first and a second sample well in fluid communication by a sample channel, and a vent channel for venting the first sample well to ambient atmosphere. An outflow channel is selectively in fluid communication between the polymerase chain reaction chamber and the first sample well. The apparatus also includes an electrophoresis separation channel orthogonal to and intersecting the sample channel, and having a first and second electrophoresis buffer well at each end thereof.
Also in accordance with the present invention, there is provided a method for performing polymerase chain reaction and capillary electrophoresis on a sample in a single, integrated device having a polymerase chain reaction portion and a capillary electrophoresis portion. A sample is injected into a polymerase chain reaction chamber. A removable barrier is adjusted to isolate the polymerase chain reaction chamber from a sample well of the capillary electrophoresis portion. The polymerase chain reaction chamber is alternately heated and cooled by performing thermal cycling. The removable barrier is adjusted to allow fluid communication between the polymerase chain reaction chamber and the first sample well. A voltage is applied across the first sample well and a second sample well in the capillary electrophoresis portion, wherein the first and second sample wells are in fluid communication via a sample channel, and a voltage is applied across first and second electrophoresis buffer wells in the capillary electrophoresis portion, wherein the first and second electrophoresis buffer wells are in fluid communication via an electrophoresis separation channel, which intersects the sample channel.